Process for production of avermectins and cultures therefor

ABSTRACT

Streptomyces avermitilis lacking branched-chain amino acid transaminase activity and/or branched-chain 2-oxo acid dehydrogenase activity, methods for preparation thereof, and use thereof to produce natural and non-natural avermectins useful as parasiticides.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08/060,949,filed May 11, 1993, now abandoned, which is a continuation ofapplication Ser. No. 07,660,912, filed Feb. 27, 1991 now U.S. Pat. No.5,238,848, which is a continuation of application Ser. No. 07/107,825,filed Oct. 13, 1987, now abandoned, which is a continuation-in-part ofapplication Ser. No. 07,006,512 filed Jan. 23, 1987, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to strains of Streptomyces avermitilis lackingbranched-chain amino acid transaminase activity and/or branched-chain2-oxo acid dehydrogenase activity, to methods for producing said S.avermitilis and to the use of S. avermitilis to produce natural andnon-natural avermectins.

2. Description of the Prior Art

U.S. Pat. Nos. 4,310,519 and 4,429,042 describe the avermectins, acomplex of related agents having potent antiparasitic activity, andtheir production by aerobic fermentation of strains of Streptomycesavermitilis; namely, S. avermitilis ATCC Nos. 31267, 31271 and 1272. Thelast two strains cited represent a frozen vial and a lyophilized tube,respectively of a culture obtained by ultraviolet irradiation of S.avermitilis ATCC 31267.

EP 214,731, published Mar. 18, 1987, the counter-part of U.S. patentapplication Ser. No. 886,867, filed Jul. 16, 1986, discloses a number ofcompounds (referred to herein as non-natural avermectins) related to thenatural or known avermectins but having a novel substituent group at the25-position, and a process for their preparation by fermentation of anavermectin producing organism in the presence of certain specifiedcarboxylic acids, or derivatives or precursors thereof. The S.avermitilis organisms used to produce the said novel C-25 substitutedavermectins are S. avermitilis ATCC 31267, 31271, 31272 and NCIB 12121.The latter organism, described in EP 214,731, is derived from S.avermitilis ATCC 31271. It gives improved yields of the novel C-25substituted avermectins when it is cultured in a semi-defined medium.Each of ATCC 31267, 31271, 31272 and NCIB 12121 may also produce, inaddition to the novel C-25 substituted derivative, varying amounts ofthe known, or natural, avermectins wherein the 25-substituent isisopropyl or (S)-sec-butyl (1-methylpropyl).

The carbon skeleton of the avermectins (depicted in formula (I) below)is derived from acetates and propionates and the C-25 substituent ofnatural avermectins from L-isoleucine (R=(S)-sec-butyl) or L-valine(R=isopropyl) [Fisher and Mrozik, "Macrolide Antibiotics", AcademicPress (1984) Ch 14].

By "known" or "natural" avermectins is meant those avermectins producedby S. avermitilis ATCC 31267, ATCC 31271 and ATCC 31272 wherein the25-position substituent is either isopropyl or(S)-sec-butyl(1-methylpropyl). Avermectins wherein the 25-positionsubstituent is other than isopropyl or sec-butyl (S-form) are referredto herein as novel or non-natural avermectins.

The strains of S. avermitilis cited in the above-mentioned U.S. patentsproduce a class of substances described generically therein as C-076.The class comprises eight distinct but closely related compoundsdescribed as C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. The "a"series of compounds refers to the natural avermectins wherein the25-substituent is (S)-sec-butyl and the "b" series to those wherein the25-substituent is isopropyl. The designations "A" and "B" refer toavermectins wherein the 5-substituent is methoxy or hydroxy,respectively. Lastly, the numeral "1" refers to avermectins wherein adouble bond is present at the 22-23 position; and numeral "2" toavermectins having a hydrogen at the 22-position and hydroxy at the 23position.

In this application no such identifiers are used as regards the25-substituent of the non-natural avermectins. Identifiers A1, A2, B1and B2 have been retained to refer to non-natural avermectins having thestructural features corresponding to those of the natural avermectins asnoted above.

Generation of mutants devoid of branched-chain alpha-keto aciddehydrogenase activity has been reported for Bacillus subtilis, Willeckeand Pardee, J. Biol. Chem. 246, 5264-72 (1971) and Pseudomonas putida,Martin et al., J. Bacteriology, 115 198-204 (1973), but not forStreptomyces.

S. avermitilis Agly-1, a mutant strain which produces virtually onlyavermectin aglycones Ala and A2a is reported by Schulman et al. J.Antibiot. 38(11), 1494-1498 (1985). Also reported is the fermentation ofS. avermitilis Agly-1 in the presence of sinefungin which causedincreased production of avermectin aglycone B components. Likewise, S.avermitilis O8, a high producing strain for avermectins, when fermentedin the presence of sinefungin as inhibitor of O-methyl transferases,resulted in production of avermectins lacking O-methyl groups on theaglycone at C-5 and in the oleandrose disaccharide moiety.

U.S. Pat. No. 4,378,353 describes C-076 related compounds and theirpreparation by cultivation of MA-5218, a mutant strain of S. avermitilisATCC 31272, obtained therefrom by ultraviolet irradiation. The mutant isidentified as ATCC 31780. The C-076 related compounds produced by saidmutant lack the C-076 furan ring. Additionally, in certain of thecompounds reported, one or both of the oleandrose sugar moieties havebeen cleaved while in others the 5-position group was oxidized to a ketogroup.

Three classes of O-methyltransferase mutants of S. avermitilis thatproduce avermectins lacking O-methyl groups have been reported by Rubyet al., 6th International Symposium on the "Biology of Actinomycetes",Debrecen, Hungary, August 26-30 (1985) and by Schulman et al,Antimicrobial Agents and Chemotherapy 31, 744-7 (1987). The first classproduces primarily B avermectins due to their inability to methylate theC-5 hydroxyl of the macrocyclic lactone ring. The second class produces3'-O, 3"-O-bis-demethylavermectins (avermectins lacking the O-methylsubstituent at the 3 position of both oleandrose monosaccharideresidues), and which are referred to as demethylavermectins. The thirdclass is unable to methylate at any position.

Schulman et al., Fed. Proc. 44, 931 (1985) disclose increased productionof B avermectins by fermenting S. avermitilis in the presence ofsubstances such as sinefungin, S-adenosylethionine andS-adenosylhomocysteine which inhibit the methylation of the C-5 hydroxygroup of the aglycone moiety by the enzyme avermectinB-O-methyltransferase. Streptomyces avermitilis mutants which lackO-methyltransferase activity and produce increased amounts of avermectinB components are also disclosed and referred to by Schulman et al. inAntimicrobial Agents and Chemotherapy 29,620-624 (1986).

Mutagenesis of S. avermitilis produces mutants which lack branched-chain2-oxo acid dehydrogenase activity or branched-chain amino-acidtransaminase activity. Mutagenesis of the thus-produced singly blockedmutants produces mutants which lack both branched-chain 2-oxo aciddehydrogenase activity and branched-chain amino acid transaminaseactivity. The mutants no longer possess the ability to producesignificant amounts of the natural avermectins in the absence of addedcompound RCOOH wherein R is isopropyl or (S)-sec-butyl, or of a compoundconvertible to RCOOH during the fermentation process. Surprisingly andunexpectedly, however, the mutants have been found to produceavermectins, natural and non-natural, when fermented in the presence ofan added compound R-COOH wherein R is isopropyl or (S)-sec-butyl, orother group disclosed herein, or of a precursor to said RCOOH. It iseven more surprising that the herein described mutants which lack onlybranched-chain 2-oxo acid dehydrogenase activity, and which are unableto degrade L-isoleucine or L-valine, are able to assimilate a widevariety of compounds into the avermectin biosynthetic pathway withproduction of non-natural avermectins free of the presence of naturalavermectins.

At least as surprising is the finding that the herein describedbranched-chain amino acid transaminase deficient mutants, which areunable to degrade L-isoleucine, L-leucine or L-valine and require thesethree amino acids in order to grow, are also able to assimilate othercompounds to produce non-natural avermectins free of the presence ofnatural avermectins.

The natural avermectins, as noted, are produced as a complex mixture ofeight distinct but closely related compounds; formula (I), R=isopropyland (S)-sec-butyl. While they have been recovered in substantially pureform (see U.S. Pat. No. 4,429,042), the methodology is, at best, 0laborious. The production of non-natural avermectins according to theprocess described in EP 214,731 may also produce some of the naturalavermectins in varying amounts due to the presence of the branched-chain2-oxo acid dehydrogenase and the amino acids L-valine and L-isoleucinein the cell of the S. avermitilis microorganisms used in theirproduction.

The ability to choose to produce either natural or non-naturalavermectins so as to minimize the number and complexity of the products,and by so doing to increase the purity of a chosen avermectin, andthereby to simplify separation procedures, is a desirable goal.

SUMMARY OF THE INVENTION

S. avermitilis strains lacking branched-chain 2-oxo acid dehydrogenaseactivity or branched-chain amino acid transaminase activity are producedby mutation of avermectin producing strains of S. avermitilis andespecially by mutation of S. avermitilis ATCC 31267, ATCC 31271, ATCC31272 or NCIB 12121. Further mutation of either of said deficientstrains produces strains deficient in both activities. The mutants areunable to synthesize the natural avermectins except where the fattyacid, or a precursor thereto, bearing the isopropyl or sec-butyl(S-form) group is added to the medium in which the mutants arefermented. They are capable of producing natural and non-naturalavermectins when fermented under aqueous aerobic conditions in anutrient medium containing an appropriate primer acid or compoundconvertible thereto in the fermentation process.

Those mutants characterized by their lack of branched-chain 2-oxo aciddehydrogenase activity, are isolated from the mutagenized colonies onthe basis of a ¹⁴ CO₂ assay. In this procedure the absence of ¹⁴ CO₂evolution by a permeabilized colony from a substrate of [¹⁴C-1]-2-oxoisocaproic acid or [¹⁴ C-1]-2-oxo-3-methyl-valeric acid or [¹⁴C-1]-2-oxo-3-methylbutyric acid indicates absence of branched-chain2-oxo acid dehydrogenase activity.

Those mutants characterized by their lack of amino acid transaminaseactivity are selected from the mutagenized colonies on the basis oftheir inability to grow on a medium which lacks L-isoleucine, L-leucineand L-valine. In practice, single colonies growing on an M9 saltsglucose-based agar medium supplemented with all the individual aminoacids found in casamino acid are transferred to a similar medium butwhich lacks L-isoleucine, L-leucine and L-valine. The herein describedmutants which are deficient in only branched-chain amino acidtransferase activity are able to use 2-oxo acids as precursors forproduction of avermectins.

It was surprising and unexpected that the herein-described mutantslacking branched-chain 2-oxo acid dehydrogenase activity and/orbranched-chain amino acid transaminase activity retained the ability toproduce avermectins, especially non-natural avermectins. The inabilityof the mutants to produce the natural fatty acyl coenzyme A derivativeswhen grown on a conventional medium could have been a lethal mutation ifmembrane integrity depended upon said derivatives or if 2-oxo acidaccumulation by the former mutant led to cytotoxicity. Furthermore,neither of the mutants were expected to be able to synthesize acetyl CoAand propionyl CoA from L-isoleucine and L-valine degradative metabolismas this requires the enzyme activities that the mutants are missing. Therequirement for these acyl CoA derivatives for avermectin biosynthesis,noted above, led to the expectation that the mutants might be severelyimpaired in non-natural avermectin production, which, surprisingly, wasnot the case.

The lack of branched-chain 2-oxo acid dehydrogenase activity in themutants described herein results in the prevention of branched-chainfatty acyl CoA synthesis from the degradation of L-isoleucine, L-leucineand L-valine and, thereby, the synthesis of the natural avermectins. Inlike manner, the branched-chain amino acid transaminase-negative mutantsof S. avermitilis also possess this characteristic lack ofbranched-chain fatty acyl CoA synthesis, and, therefore, the inabilityto produce the natural avermectins. This lack of fatty acyl CoA is dueto two reasons. First, such transaminase-negative mutants are not ableto synthesize branched-chain 2-oxo acids from medium-suppliedisoleucine, leucine and valine via the normal route of transamination.Secondly, in these transaminase mutants, branched-chain 2-oxo acidproduction by the cellular branched-chain amino acid biosyntheticpathway is prevented by the necessary inclusion of these amino acids inthe fermentation growth medium. The presence of these amino acidsprevents operation of this biosynthetic pathway (and production of theintermediate 2-oxo acids) by well-known mechanisms of enzyme repressionand feed-back inhibition by these amino acid end products of thepathway. The unavailability of these 2-oxo acids, which are substratesfor the active branched-chain 2-oxo acid dehydrogenase enzyme,effectively prevents branched-chain fatty acyl CoA synthesis. Thus, thepresent invention encompasses the use of such 2-oxo acid dehydrogenasenegative and transaminase-negative mutants, and mutants in which boththe branched-chain transaminase negative and 2-oxo aciddehydrogenase-negative mutations are combined.

The present invention also includes any organism, regardless of itsappearance or physiological behavior, that may be developed by means oftransformation, transduction, genetic recombination or some othergenetical procedure, using a nucleic acid or an equivalent material fromthe herein described species, whereby it has acquired thecharacteristics of the herein described mutants.

The terms "avermectin" or "avermectins" as used herein refers tocompounds having formula (I) below but wherein the 25-substituent (R)can be any group assimilable at said position by the S. avermitilis ofthis invention.

The herein described mutants are highly valuable for producingnon-natural avermectins by the processes disclosed and exemplifiedherein. They are especially valuable for production of preferredavermectins, i.e., compounds wherein the C-25 substituent is C₄ -C₆cycloalkyl or cycloalkenyl, optionally substituted by C₁ -C₄ alkylgroup; 1-methylthioethyl, or a 5- or 6-membered oxygen or sulfurheterocyclic group, especially 3-thienyl or 3-furyl.

DETAILED DESCRIPTION OF THE INVENTION

Mutation of an avermectin producing member of the species Streptomycesavermitilis is carried out according to known procedures using any of avariety of mutating agents including ultraviolet irradiation, X-rayirradiation, N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonate, nitrous acid and nitrogen mustards, e.g.,N-methylbis(2-chloroethyl)amine, or like treatments. The mutagenesis canbe conducted on spores or on a vegetative culture of S. avermitiliscapable of producing natural avermectins, e.g., S. avermitilis ATCC31272.

Following procedures well known to those skilled in the art, mutagenizedcolonies are selected for lack of branched-chain 2-oxo aciddehydrogenase on the basis of a biochemical assay method which permitsscreening of large numbers of randomly mutagenized bacterial coloniesfor ¹⁴ CO₂ production from [¹⁴ C-1]-2-oxo acids (Tabor et al., J. Bact.128, 485-486, 1976).

The methodology comprises growing the mutant colonies in the wells of amicrotiter plate on a suitable nutrient medium, permeabilizing the cellswith toluene followed by adding the [¹⁴ C-1]-2-oxo acid (e.g.2-oxoisocaproic acid) to each well and checking the atmosphere above thefermentation for ¹⁴ CO₂. Alternatively, [¹⁴ C-1]-2-oxo-3-methylvalericacid, or [¹⁴ C-1]-2-oxo-3-methylbutyric acid can be used in place of [¹⁴C-1]-2-oxo-isocaproic acid. Production of ¹⁴ CO₂ is conveniently checkedfor by placing moist Ba(OH)₂ -saturated filter paper above theindividual wells to trap any ¹⁴ CO₂ released and detection of Ba¹⁴ CO₃,if any, by autoradiography. Mutants which lack branched-chain 2-oxo aciddehydrogenase activity give autoradiograms approximating those of blankcontrols; i.e., no Ba¹⁴ CO₃ is produced by the mutants.

The mutants thus obtained are subjected to further mutagenesis using anyof the above-mentioned mutating agents. Mutagenesized colonies areselected for lack of branched-chain amino acid transaminase activity onthe basis of their failure to grow on M9/glucose minimal plates exceptin the presence of L-isoleucine, L-leucine and L-valine (ILV). All threeamino acids must be present for growth to occur. Further, it has beendemonstrated that said transaminase negative mutants do not grow onmedia supplemented with all three of the keto acids which serve assubstrates for the transaminase reactions. A single transaminase enzymethus catalyzes transamination of each of the three keto acids(2-oxo-3-methyl valeric acid, 2-oxo-isocaproic acid, 2-oxo-isovalericacid).

The doubly blocked mutants, those lacking both branched-chain 2-oxo aciddehydrogenase and branched-chain amino acid transaminase, activities areof particular interest since the probability of their reverting tocultures which produce the natural avermectins is extremely low. Thesingly blocked mutants may, under certain circumstances, revert tocultures which would produce natural avermectins.

In addition to production of desired alleles of a given strain ofmicroorganism by mutagenesis, protoplast fusion permits introduction ofdesirable alleles produced/identified in one strain into the chromosomeof another strain. For example, a strain of S. avermitilis deficient inbranched-chain 2-oxo acid dehydrogenase activity and branched-chainamino acid transaminase activity can, by protoplast fusion with a S.avermitilis strain having the aforementioned activities produce a strainof S. avermitilis deficient only in branched-chain amino acidtransaminase activity. As those skilled in the art recognize, protoplastfusion technology enables combination of desirable alleles fromdivergent lines of selection into a single strain. The herein describedS. avermitilis JC-923 (ATCC 53669), a branched-chain amino acidtransaminase deficient strain was produced via this technology.

The morphological and cultural characteristics of the mutants of thisinvention are generally as described in U.S. Pat. No. 4,429,042. Thedistinguishing characteristic of the mutants of this invention is theirlack of branched-chain 2-oxo acid dehydrogenase activity and/or ofbranched-chain amino acid transaminase activity which characteristicsare determined as described herein. The lack of said activities resultsin the failure of the mutants to produce the natural avermectins whengrown on a defined medium substantially free of fatty acids RCOOHwherein R is isopropyl or (S)-sec-butyl, or compounds convertible tosaid RCOOH during fermentation. A taxonomic investigation conducted bythe American Type Culture Collection, confirmed that the characteristicsof two mutant strains I-3 and HL-026, selected by the above ¹⁴ CO₂assay, bear a close relationship to those of the parental ATCC 31272strain described in U.S. Pat. No. 4,429,042, but with certainexceptions. Thus, mutant strain I-3 (ATCC 53567) forms significantlyfewer spore chains than does ATCC 31272, and mutant strain HL-026 (ATCC53568) is practically devoid of aerial mycelia and spores, but the veryfew spore chains it produces are of similar character to those of ATCC31272. Also, mutant HL-026 exhibits a doubtful capacity to utilizeraffinose as a sole carbon source, whereas the ATCC 31272 strain andmutant I-3 strain are able to use raffinose. (In experiments byapplicants, raffinose did not appear to support the growth of any ofthese strains). One further characteristic of mutant strain HL-026 wasthat it produced less melanin pigment than the other two strains anduniquely none at all on tyrosine agar. Finally, in contrast to thedescription given for ATCC 31272 in U.S. Pat. No. 4,429,042, we areunable to detect growth of the mutants or of ATCC 31272 with sucrose assole carbon source. Mutants I-3 and HL-026 are deficient only inbranched-chain 2-oxo acid dehydrogenase activity. The doubly deficientmutant PGS-119 (ATCC 53670), produced by further mutagenesis of mutantI-3 (ATCC 53567), and JC-923 (ATCC 53669), obtained by protoplastfusion, bears a similar taxonomic relation to ATCC 31272 as does mutantstrain I-3.

Streptomyces avermitilis I-3, HL-026, PGS-119 and JC-923 have beendeposited under the terms of the Budapest Treaty in the American TypeCulture Collection, Rockville, Md., a recognized depository affordingpermanence of the deposits and ready accessibility thereto by the publicif a patent is granted on this application. They have been given thedesignation Streptomyces avermitilis ATCC 53567, ATCC 53568, ATCC 53670and ATCC 53669, respectively. The deposits are available during pendencyof this application to one determined by the Commissioner of the UnitedStates Patent and Trademark Office to be entitled thereto under 37 CFR1.14 and 35 USC 122, and in accordance with foreign patent laws incountries wherein counterparts of this application, or its progeny, arefiled. All restrictions on the availability to the public of themicroorganisms deposited will be irrevocably removed upon granting ofthe patent.

Each of S. avermitilis ATCC 31267, ATCC 31271, ATCC 31272 and NCIB 12121produces the natural avermectins, formula (I) compounds ##STR1## whereinthe broken line at the 22-23 position represents an optional doublebond;

R¹ is hydroxy and is present only when the double bond is absent;

R² is 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy of the formula##STR2## R³ is hydrogen or methyl; and R is isopropyl or (S)-sec-butyl.U.S. Pat. No. No. 4,285,963 describes an avermectin of formula (I)wherein the 25-position is substituted with a methyl and an ethyl group;R¹ is hydroxy and R³ is methyl.

In the non-natural avermectins referred to herein R is a substituentother than isopropyl or (S)-sec-butyl and is as defined below.

The compounds essential for utilization in the biosynthesis of formula(I) compounds occur in the cell of S. avermitilis. These compounds,L-valine, L-leucine and L-isoleucine, are believed to enter into thebiosynthesis of avermectins via conversion to 2-oxo acid anddecarboxylation of the acid by branched-chain 2-oxo acid dehydrogenase,concomitant with coupling the product with coenzyme A. Their presenceaccounts for the concurrent production of both the isopropyl and(S)-sec-butyl compounds of formula (I). This, of course, gives rise toproblems in separating the isopropyl from the (S)-sec-butyl derivatives.

When fermented in a nutrient medium containing the appropriate primercompound the mutants of this invention produce a compound of formula (I)or, as is more usually the case, a mixture of two or more compounds offormula (I) in which R corresponds to the primer compound used. Up tofour products, conveniently and trivially referred to as R-avermectinA1, A2, B1 and B2, according to the designations used in U.S. Pat. No.4,429,042, can be produced. The "R-" group, of course, refers to theC-25 substituent. For example, when R is cyclopentyl the four possibleavermectins are:

    ______________________________________                                        Trivial Name     R.sup.1       R.sup.3                                        ______________________________________                                        cyclopentyl      double bond   CH.sub.3                                       avermectin A1                                                                 cyclopentyl      hydroxy       CH.sub.3                                       avermectin A2                                                                 cyclopentyl      double bond   H                                              avermectin B1                                                                 cyclopentyl      hydroxy       H.                                             avermectin B2                                                                 ______________________________________                                    

In the non-natural avermectins the C-25 substituent "R" of formula (I)is other than isopropyl or (S)-sec-butyl.

Compounds of formula (I) wherein the double bond is present and OH isabsent may alternatively be prepared from the corresponding compound offormula (I) wherein R¹ is OH and the double bond is absent by adehydration reaction. The reaction is performed by first selectivelyprotecting the hydroxy groups at the 5 and 4" positions, e.g. as thet-butyldimethylsilyloxy acetyl derivative, then reacting with asubstituted thiocarbonyl halide, such as (4-methylphenoxy)thiocarbonylchloride, followed by heating in a high boiling point solvent, e.g.trichlorobenzene, to effect the dehydration. The product is finallydeprotected to give the unsaturated compound. These steps together withappropriate reagents and reaction conditions are described in U.S. Pat.No. 4,328,335.

Formula (I) compounds wherein R³ is H may also be prepared from thecorresponding compounds wherein R³ is CH₃ by demethylation. Thisreaction is achieved by treating the 5-methoxy compound, or a suitablyprotected derivative thereof, with mercuric acetate and hydrolyzing theresulting 3-acetoxy enol ether with dilute acid to give the 5-ketocompound. This is then reduced using, for example, sodium borohydride toyield the 5-hydroxy derivative. Appropriate reagents and reactionconditions for these steps are described in U.S. Pat. No. 4,423,209.

Compounds of formula (I) wherein R¹ is H and the double bond is absentcan be prepared from the corresponding compound wherein the double bondis present and R¹ is absent, by selective catalytic hydrogenation usingan appropriate catalyst. For example, the reduction may be achievedusing tris(triphenylphosphine)rhodium (I) chloride as described inEuropean Patent Application Publication No. 0001689, and its counterpartU.S. Pat. No. 4,199,569, issued Apr. 22, 1980.

The compounds of formula (I) wherein R² is H are prepared from thecorresponding compounds wherein R² is4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy by removing the4'-(alpha-L-oleandrosyl)-alpha-L-oleandrose group by mild hydrolysiswith an acid in an aqueous organic solvent to yield the aglycone havinga hydroxy group at the 13-position; this is then halogenated, forexample by reaction with a benzene sulfonyl halide, to yield the13-deoxy-13-halo derivative which is finally selectively reduced, forexample using tributyltin hydride. In order to avoid unwanted sidereactions it is desirable to protect any other hydroxy groups which maybe present, for example using a tert-butyldimethylsilyl group. This isthen readily removed after the halogenation or reduction step bytreatment with methanol containing a trace of acid. All these stepstogether with appropriate reagents and reaction conditions for theirperformance are described in European Patent Application Publication No.0002615.

The compounds capable of utilization by the S. avermitilis of thisinvention for the biosynthesis of avermectins, natural and non-natural,are compounds of formula (II-A)

    R--COOH                                                    (II-A),

including compounds convertible to (II-A) during the fermentationprocess. Said compounds are referred to herein as "primer compounds". Informula (II-A), R is an alpha-branched-chain group, the carbon atomthereof to which is attached the -COOH group is also attached to atleast two other atoms or groups other than hydrogen. This definition, ofcourse, embraces saturated and unsaturated acyclic and cyclic groups,including those optionally bearing a sulfur or oxygen heteroatom as amember of the acyclic chain or cyclic ring.

More specifically, R, which becomes the C-25 substituent, can be analpha-branched C₃ -C₈ alkyl, alkenyl, alkynyl, alkoxyalkyl oralkylthioalkyl group; a C₅ -C₈ cycloalkylalkyl group wherein the alkylgroup is an alpha-branched C₂ -C₅ alkyl group; a C₃ -C₈ cycloalkyl or C₅-C₈ cycloalkenyl group, either of which may optionally be substituted bymethylene or one or more C₁ -C₄ alkyl groups or halo atoms (fluoro,chloro, iodo or bromo); or a 3 to 6 membered oxygen or sulfur containingheterocyclic ring which may be saturated, or fully or partiallyunsaturated and which may optionally be substituted by one or more C₁-C₄ alkyl groups or halo atoms.

Compounds convertible to RCOOH; i.e., precursors, in the fermentationprocess are compounds of formulae (II-B) wherein R is as defined above:

    R--(CH.sub.2).sub.n --Z                                    (II-B)

n is 0, 2, 4 or 6; and Z is --CH₂ OH, --CHO, --CH₂ NH₂, --COOR⁵ or--CONHR⁶ wherein R⁵ is H or (C₁₋₆)alkyl; R⁶ is hydrogen, (C₁₋₄)alkyl, orthe residue of an amino acid, especially of aspartic acid, glutamic acidand methionine, e.g., --CH(COOH)CH₂ COOH, --CH(COOH)(CH₂)₂ COOH and--CH(COOH)(CH₂)₂ SCH₃, respectively.

In the case of S. avermitilis strains deficient only in branched-chainamino acid transaminase, 2-oxo acids also serve as precursors. Thus, forsaid strains acids of the formula (II-C)

    R--CO--Z                                                   (II-C)

wherein R and Z are as defined above are capable of utilization by saidS. avermitilis for the biosynthesis of avermectins.

Also included in this invention are the isomeric forms of formula (II-A)compounds, and compounds convertible thereto during the fermentationprocess, and the isomeric avermectins at C-25 resulting from their usein the herein described process.

The process of this invention is carried out by aerobically fermentingwith a strain of S. avermitilis which lacks branched-chain 2-oxo aciddehydrogenase activity and/or branched-chain amino acid transaminaseactivity in an aqueous nutrient medium comprising an assimilable sourceof nitrogen, carbon, inorganic salts and a compound of formula RCOOH, ora compound convertible to said compound (i.e., a precursor) during thefermentation. The acid, or compound convertible thereto, is added to thefermentation either at the time of inoculation or at intervals duringthe fermentation. When a transaminase negative mutant is used, themedium must contain L-isoleucine, L-leucine and L-valine in order forgrowth of the mutant to occur. Production of the avermectin products maybe monitored by removing samples from the fermentation, extracting withan organic solvent and following the appearance of the product bychromatography, for example, using high performance liquidchromatography. Incubation is continued until the yield of the producthas been maximized, generally for a period of from 4 to 15 days.

A preferred level of each addition of the primer compounds (carboxylicacid or compound convertible thereto) is between 0.05 and 3.0 grams perliter. The primer compound can be added continuously, intermittently orall at once to the fermentation. The acid (RCOOH) is added as such or asa salt, such as the sodium, lithium or ammonium salt, or as a compoundconvertible to the acid as defined above. The acid, if a solid, ispreferably dissolved in a suitable solvent such as water or(C₁₋₄)alcohols.

The media used for the fermentation can, especially when the C-25substituent is to be isopropyl or (S)-sec-butyl, be conventional mediacontaining assimilable sources of carbon, nitrogen and trace elements.When the C-25 substituent is to be a non-natural group; i.e., it is notisopropyl or (S)-sec-butyl, the fermentation medium is one in which thechosen ingredients lack, or contain only minimal amounts of primercompounds wherein the R moiety is isopropyl or (S)-sec-butyl.

After fermentation for a period of several days at a temperaturepreferably in the range of 24° to 33° C., the fermentation broth iscentrifuged or filtered and the mycelial cake is extracted withpreferably acetone or methanol. The solvent extract is concentrated andthe desired product is then extracted into a water-immiscible organicsolvent, such as methylene chloride, ethyl acetate, chloroform, butanolor methyl isobutyl ketone. The solvent extract is concentrated and thecrude product is further purified as necessary by chromatography, forexample using preparative reverse phase, high performance liquidchromatography.

The product is generally obtained as a mixture of the compounds offormula (I) wherein R² is4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy, R¹ is OH and the doublebond absent or R¹ is absent and the double bond is present and whereinR³ is H or CH₃ ; however, the proportions can vary depending on theparticular mutant and primer compound employed and the conditions used.

The source of the R group; i.e., whether it comes directly from R-COOHor is produced from one of the above precursors, or from any precursor,is immaterial to the production of the avermectins. The criticalrequirement of the process of this invention for their production isthat the desired R group be made available to the S. avermitilis strainsof this invention in the fermentation process.

Suitable compounds include the following:

2,3-dimethylbutyric acid

2-methylhexanoic acid

2-methylpent-4-enoic acid

2-cyclopropyl propionic acid

4,4-difluorocyclohexane carboxylic acid Lithium salt

4-methylenecyclohexane carboxylic acid

3-methylcyclohexane carboxylic acid (cis/trans)

1-cyclopentene carboxylic acid

1-cyclohexene carboxylic acid

tetrahydropyran-4-carboxylic acid

thiophene-2-carboxylic acid

3-furoic acid

2-chlorothiophene-4-carboxylic acid

cyclobutane carboxylic acid

cyclopentane carboxylic acid

cyclohexane carboxylic acid

cycloheptane carboxylic acid

2-methylcyclopropane carboxylic acid

3-cyclohexene-1-carboxylic acid

2-methylthiopropionic acid

2-methyl-4-methoxybutyric acid

thiophene-3-carboxylic acid

hydroxymethylcyclopentane

3-thiophene carboxaldehyde

3-cyclohexylpropionic acid

3-cyclopentylpropionic acid

hydroxymethylcyclobutane

tetrahydrothiophene-3-carboxylic acid

3-cyclopentyl-1-propanol

3-methylcyclobutane carboxylic acid Lithium salt

3-fluorocyclobutane carboxylic acid

3-methylenecyclobutane carboxylic acid Lithium salt

2-methyl-4-methylthiobutyric acid

tetrahydrothiopyran-4-carboxylic acid

cyclobutylmethylamine

ethyl cyclobutanecarboxylate

4-hydroxymethylcyclopentene

2-(3-thiophenecarbonyl) propionic acid ethyl ester

(S)-2-methylpentanoic acid

(R)-2-methylpentanoic acid

O-methyltransferase mutants can be obtained from the herein-describedbranched-chain 2-oxo acid dehydrogenase negative mutants and/orbranched-chain amino acid transaminase negative mutants. Mutants inwhich a mutation in active branched-chain 2-oxo acid dehydrogenaseactivity and/or branched-chain amino acid transaminase activity iscombined with one or both of the O-methyltransferase mutations yieldstrains of S. avermitilis that will, when fed RCOOH compounds orcompounds convertible to RCOOH during the fermentation process, produceprimarily B avermectins, demethyl-avermectins or demethylavermectin Bcompounds. Said mutants are obtained by mutagenesis of the hereindescribed mutants which lack branched-chain 2-oxo acid dehydrogenaseactivity and/or branched-chain amino acid transaminase activity by meansof ultraviolet light and/or chemical mutagens such asN-methyl-N-nitrosourethan, nitrosoguanidine, ethyl methane sulfonate orother agent such as those enumerated above. Alternatively,branched-chain 2-oxo acid dehydrogenase positive mutants and/orbranched-chain amino acid transaminase positive mutants which lack oneor both of the O-methyltransferases can be mutated by treatment with UVlight or a mutagenizing agent to produce the branched-chain 2-oxo aciddehydrogenase negative mutants and/or branched-chain amino acidtransaminase negative mutants.

The non-natural avermectins produced by such mutants are characterizedby the presence of hydroxy groups at the C-5 position of the aglyconemoiety and/or the C-3' and/or C-3" positions of the oleandrose moieties.

The above-described mutants are identified according to the methodologydescribed by Schulman et al. Antimicrobial Agents and Chemotherapy, 29,620-624 (1986). They are useful for the same purposes and in the sameway as are the known avermectins.

Alternatively, increased amounts of the B avermectins, including thoselacking methyl groups on the oleandrose disaccharide moiety, areproduced by fermenting the mutants of this invention, which lack activebranched-chain 2-oxo acid dehydrogenase and/or branched-chain amino acidtransaminase activity, in the presence of a substance such assinefungin, S-adenosyl-ethionine or S-adenosylhomocysteine whichinhibits O-methyl transferase activity.

The compounds of the invention are highly active antiparasitic agentshaving particular utility as anthelmintics, ectoparasiticides,insecticides and acaricides.

Thus the compounds are effective in treating a variety of conditionscaused by endoparasites including, in particular, helminthiasis which ismost frequently caused by a group of parasitic worms described asnematodes and which can cause severe economic losses in swine, sheep,horses and cattle as well as affecting domestic animals and poultry. Thecompounds are also effective against other nematodes which affectvarious species of animals including, for example, Dirofilaria in dogsand various parasites which can infect humans includinggastro-intestinal parasites such as Ancylostoma, Necator, Ascaris,Strongyloides, Trinchinella, Capillaria, Trichuris, Enterobius andparasites which are found in the blood or other tissues and organs suchas filiarial worms and the extract intestinal states of Strongyloidesand Trichinella.

The compounds are also of value in treating ectoparasite infectionsincluding in particular arthropod ectoparasites of animals and birdssuch as ticks, mites, lice, fleas, blowfly, biting insects and migratingdipterous larvae which can affect cattle and horses.

The compounds are also insecticides active against household pests suchas the cockroach, clothes moth, carpet beetle and the housefly as wellas being useful against insect pests of stored grain and of agriculturalplants such as spider mites, aphids, caterpillars and against migratoryorthopterans such as locusts.

The compounds of formula (I) are administered as a formulationappropriate to the specific use envisaged and to the particular speciesof host animal being treated and the parasite or insect involved. Foruse as an anthelmintic the compounds may be administered orally in theform of a capsule, bolus, tablet or a liquid drench, or alternatively,they may be administered by injection or as an implant. Suchformulations are prepared in a conventional manner in accordance withstandard veterinary practice. Thus capsules, boluses or tablets may beprepared by mixing the active ingredient with a suitable finely divideddiluent or carrier additionally containing a disintegrating agent and/orbinder such as starch, lactose, talc, magnesium stearate etc. A drenchformulation may be prepared by dispersing the active ingredient in anaqueous solution together with dispersing or wetting agents, etc., andinjectable formulations may be prepared in the form of a sterilesolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. These formulationswill vary with regard to the weight of active compound depending on thespecies of host animal to be treated, the severity and type of infectionand the body weight of the host. Generally for oral administration adose of from about 0.001 to 10 mg per kg of animal body weight given asa single dose or in divided doses for a period of from 1 to 5 days willbe satisfactory, but, of course, there can be instances where higher orlower dosage ranges are indicated and such are within the scope of thisinvention.

As an alternative the compounds may be administered with the animalfeedstuff and for this purpose a concentrated feed additive or premixmay be prepared for mixing with the normal animal feed.

For use as an insecticide and for treating agricultural pests thecompounds are applied as sprays, dusts, emulsions and the like inaccordance with standard agricultural practice.

Production of Branched-chain 2-oxo Acid Dehydrogenase Deficient S.avermitilis I-3 (ATCC 53567)

Step 1. S. avermitilis ATCC 31272 was grown as a confluent lawn on NewPatch Agar Medium for 12 days at 30° C. The medium comprised

    ______________________________________                                        V-8 Juice*           200     ml                                               CaCO.sub.3           3       grams                                            Agar                 15      grams                                            H.sub.2 O            to 1000 ml                                               Nutrient broth       1.0     grams/L                                          sodium acetate.3H.sub.2 O                                                                          1.4     grams/L                                          isovaleric acid      50      mg/L                                             isobutyric acid      50      mg/L                                             2-methylbutyric acid 50      mg/L                                             isoleucine           250     mg/L                                             leucine              250     mg/L                                             valine               250     mg/L                                             trace elements solution**                                                                          1       ml/L                                             *A mixture of 8 vegetable juices (tomato, carrots, celery, beets,             parsley, lettuce, watercress and spinach) plus salt, ascorbic and             citric acids and natural flavors. Available from Campbell Soup                Company, Camden, NJ.                                                          **Composition of Trace elements solution:                                     FeCl.sub.3.6H.sub.2 O                                                                   2.7 g                                                               MnSO.sub.4.H.sub.2 O                                                                    4.2                                                                 CuSO.sub.4.5H.sub.2 O                                                                   0.5                                                                 CaCl.sub.2                                                                              11.0                                                                H.sub.3 BO.sub.3                                                                        0.62                                                                CoCl.sub.2.6H.sub.2 O                                                                   0.24                                                                ZnCl.sub.2                                                                              0.68                                                                Na.sub.2 MoO.sub.4                                                                      0.24                                                                ______________________________________                                    

Dissolve the above in 1 liter of 0.1N HCl.

Spores were harvested from 3 such plates and suspended in 20 ml. of0.05M tris-maleic acid buffer, pH 9.0.

Step 2. 10 ml of the spore suspension was added to a vial containing 10mg of N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The vial was incubatedand shaken at 28° C. for 60 minutes and the spores then washed profuselywith 1% NaCl solution.

Step 3. The washed spores were suspended in 1% NaCl and mixed with anequal volume of 80% ethylene glycol. This suspension was preserved at-20° C. and used as a source of cells to be screened for mutants. Itgave approximately 10⁴ colonies/ml when germinated.

This spore stock was spread on YPD plates to yield approximately 100colonies per plate (YPD medium comprises 10 g/l of each of yeastextract, Bacto peptone* and dextrose; and 15 g/l of Bacto agar*,adjusted to pH 6.9 before autoclaving) Ingredients marked with anasterisk are available from Difco Laboratories, Detroit, Mich. 48238.

Step 4. Single colonies were picked from plates after 2-3 weeks ofgrowth at 28° C. and placed in individual wells of a standard 96 wellmicrotiter plate. Also, a small quantity of the colony was patched ontoa fresh agar medium to serve as a source of viable cells when mutantsare identified.

Step 5. To each well was added approximately 75 microliters of a liquidM9 salts medium containing 1% glucose, 0.1% casamino acids, and 0.01% ofeach of isovaleric, isobutyric and 2-methylbutyric acids. After severaldays of incubation at 28° C., the cells were assayed for the presence ofbranched-chain 2-oxo acid dehydrogenase. (Each liter of M9 salts mediumcomprises 6 g Na₂ HPO₄, 3 g KH₂ PO₄, 0.5 g NaCl and 1 g of NH₄ Cl. Themedium is autoclaved and then 1 ml of each of sterilized 1M MgSO₄ and0.1M CaCl₂ are added aseptically).

Step 6. A microsuspension of 5% toluene in M9 salts medium was preparedby a brief sonication of the immiscible mixture. To 25 ml of thissuspension was added 1.2 ml of a solution containing [¹⁴C-1]-2-oxo-isocaproic acid, 2.5 microcurie/ml and 10.0microcurie/micromole. 50 Microliters of this overall mixture was addedto each of the wells of the microtiter plates containing the colonies tobe assayed.

Step 7. The ¹⁴ CO₂ produced from each well was trapped and visualized bythe procedure described by Tabor et al., J. Bacteriol. 128 485-486(1976) entitled "Convenient Method for Detecting ¹⁴ CO₂ in MultipleSamples: Application to Rapid Screening for Mutants". Mutants lackingactive branched-chain 2-oxo acid dehydrogenase produce no Ba¹⁴ CO₃beyond that observed for the controls.

A more refined method which improves the contrast between a positiveassay for ¹⁴ CO₂, indicated by a dark spot on the autoradiogram as aresult of Ba¹⁴ CO₃ formation, and a negative assay indicated by no spotor a very light spot, comprises the following modified screen.

Single colonies (see Step 4 above) were picked from the agar mediumafter 7-14 days of growth (rather than 2-3 weeks and assayed directly bysteps 6 and 7 above). Step 5 of the above procedure is omitted.

An even more refined assay method which is quantitative in nature asregards ¹⁴ CO₂ release comprises growing the mutants detected by theabove screens on a suitable medium comprising M9 salts medium withglucose, 1% and "Syncasa-bcaa", 0.1% (a synthetic mixture of L-aminoacids with the approximate composition of commercial casamino acids, butwithout the presence of L-valine, L-isoleucine and L-leucine, seebelow).

After growth to high cell density, the cells were washed in M9 saltsmedium and resuspended in cold M9 salts medium containing 1% toluenewhich had been sonicated to produce a milky white dispersion of thetoluene. The cell/buffer/toluene suspension was incubated for 40 minutesat 30° C. in order to permeabilize the cells. The permeabilized cellswere then washed in M9 medium salts and finally resuspended in one-fifththe original volume of M9 medium buffer. 180 Microliters of thissuspension were used per assay.

A reaction volume of 300 microliters contained the toluenized cells,thiamine pyrophosphate (TPP), 0.4 mM; coenzyme A (CoA), 0.11 mM;nicotinamide adenine dinucleotide (NAD), 0.68 mM, dithiothreitol (DTT),2.6 mM; MgCl₂, 4.1 mM; Tris-HCl, 60 mM; Tris-HCl, 60 mM, pH 7.5; and [¹⁴C-1]-alpha-ketoisocaproate, 6,000 cpm, microcurie per micromole. Theefficiency of counting was 73%. The reaction was carried out in 15 mlscintillation vials containing a 2×2 cm Whatman #4 paper square pressedinto the screw cap of the vial. The paper contains 30 microliters of 1MHyamine Hydroxide (1M solution of methylbenzethonium hydroxide inmethanol; available from Sigma Chemical Co., St. Louis, Mo. 63178),which traps ¹⁴ CO₂ evolved in the reaction. After incubation for 2hours, the papers are immersed in 10 ml of Beckman Aquasol II (UniversalLSC (liquid scintillation counter) available from New England NuclearResearch Products, Boston, Mass. 02118) and the radioactivity measuredin a liquid scintillation counter after equilibration in this solventfor 4 hours or more. A blank control reaction (i.e.--no cells) gives ca.50-300 cpm.

Mutant I-3 and others like it gave counts that were less than or equalto the blank control reaction, whereas the parent strain gave countsseveral fold higher than the blank control value.

Isolation of HL-026 Derivative (ATCC 53568) of S. Avermitilis I-3 (ATCC53567)

S. avermitilis I-3 (ATCC 53567) was streaked out on nutrient agarplates. A relatively high frequency of spontaneous variants appeared,some of which lacked aerial mycelium upon 4 days incubation at 30° C.Several of such variants were isolated and tested for their ability toproduce non-natural avermectins when fermented in AP-5 medium to whichcyclopentane carboxylic acid was added. From the isolates, many of whichproduced non-natural avermectins free of natural avermectins, a strainwhich yielded higher titers of avermectins in flask experiments than itsparent S. avermitilis I-3 (ATCC 53567) was assigned identificationnumber HL-026 (ATCC 53568).

Production of Branched-chain 2-oxo Acid Dehydrogenase Deficient andBranched-chain Amino Acid Transaminase Deficient S. avermitilis PGS-119(ATCC 53670)

Step 1. Approximately 100 mg of S. avermitilis I-3 (ATCC 53567), grownon a fresh SAMM agar plate for four days, was inoculated into a 300 mlflask containing 50 ml of SCM medium (pH 7.2). The flask was then shakenat 200 RPM and 30° C. for twenty-four hours (final pH=8.2).

Step 2. The flask was removed from the shaker and 10 ml of the wholebroth centrifuged in a sterile tube for five minutes at 2000 RPM. Thecells were then resuspended in 50 ml of SCM medium in sterile 300 ml.Erlenmeyer flasks and the flasks shaken on a rotary shaker for two hoursat 30° C.

Step 3. The 10 ml of the suspension was placed in a sterile tube.

Step 4. Ethylmethane sulfonate (250 μl) was added to the tube (in a wellventilated hood), the contents thoroughly mixed, then poured into asterile 300 ml flask and the flask shaken in a rotary shaker for threehours at 30° C.

Step 5. Fresh sterile SCM medium (40 ml) was added to the flask andshaking continued for a total of 70 hours at 30° C.

Step 6. The flask was removed, the contents spun down at 8000 RPM forten minutes at 20° C. The cells were washed by re-suspending in SCMmedium, spun down again and re-suspended in 10 ml SCM medium.

Step 7. Cells were removed and assayed via replica plating, ca. 150colonies/plate, for their ability to grow on M9/glucose minimal platesin the presence and absence of L-leucine, L-isoleucine, L-valine andcombination of any of said amino acids. The mutant cells of interestgrew only on media supplemented with L-leucine, L-isoleucine andL-valine. These derivatives of S. avermitilis I-3 (ATCC 53567),deficient in branched-chain amino acid transaminase activity, alsofailed to grow on media supplemented with one or more of the three 2-oxoacids (2-oxoisocaproic acid; 2-oxo-3-methylvaleric acid and2-oxoisovaleric acid) which serve as precursors for L-leucine,L-isoleucine and L-valine. This behavior is completely opposite to thatof S. avermitilis I-3 (ATCC 53567) which grew well on such media. Thus,a single transaminase enzyme catalyzes transamination of said 2-oxoacids.

    ______________________________________                                        SCM MEDIUM                                                                    ______________________________________                                        Yeast autolysate         10 g/l                                               Beef extract             5 g/l                                                Casein enzymatic hydrolysate                                                                           10 g/l                                               1M MgSO.sub.4            3 g/l                                                1M K.sub.2 HPO.sub.4 ; pH 7.0 (HCl)                                                                    100 g/l                                              ______________________________________                                        SAMM Agar Plate                                                                                g/L                                                          ______________________________________                                               Na.sub.2 HPO.sub.4                                                                      6.0                                                                 KH.sub.2 PO.sub.4                                                                       3.0                                                                 NaCl      0.5                                                                 NH.sub.4 Cl                                                                             1.0                                                                 1M MgSO.sub.4                                                                           1.0                                                                 0.1M CaCl.sub.2                                                                         1.0                                                                 Dextrose  8.0                                                                 Casamino Acids                                                                          20.0                                                                Agar      20.0                                                         ______________________________________                                        Composition of "Syncasa - bcaa", 100 fold Concentrate                                         grams/liter                                                   ______________________________________                                        L-alanine       3                                                             L-arginine      4                                                             L-aspartic acid 6                                                             L-cystine       1                                                             L-glutamic acid 20                                                            glycine         1                                                             L-histidine     2                                                             L-lysine        7                                                             L-methionine    3                                                             L-phenylalanine 6                                                             L-proline       10                                                            L-serine        6                                                             L-threonine     4                                                             L-tyrosine      4                                                             L-tryptophan    1                                                             ______________________________________                                    

The mixture is adjusted to pH 7 and filter sterilized. One volume ofconcentrate is added to 99 volumes of medium to achieve standard useconcentrations.

S. avermitilis JC-923 (ATCC 53669) by Protoplast Fusion of aSpectinomycin Resistant Strain of S. avermitilis ATCC 31272 and S.avermitilis PGS 119 (ATCC 53670).

S. avermitilis ATCC 31272 spectinomycin resistant, is a spontaneousmutant of S. avermitilis ATCC 31272. It was isolated from populations ofvegetative mycelia of ATCC 31272 spread on AS-1 agar plates containing50 mcg/ml of spectinomycin. Spores of the mutant germinated on richmedium are resistant to 50 mcg/ml spectinomycin, as compared to sporesof the isogenic parental strain which fail to germinate under theseconditions. This dominant selectable marker was used successfully toisolate branched-chain amino acid transaminase-deficient isolates ofATCC 31272.

    ______________________________________                                        AS-1 agar                                                                     (Rich plating medium for Streptomyces)                                        ______________________________________                                        Yeast extract         1     g                                                 L-Alanine             0.2   g                                                 L-Arginine            0.2   g                                                 L-Asparagine          0.5   g                                                 Soluble starch        5     g                                                 NaCl                  25    g                                                 Na.sub.2 SO.sub.4     10    g                                                 Agar                  20    g                                                 Distilled water       1     liter                                             ______________________________________                                    

PROCEDURES TO PRODUCE VIABLE PROTOPLASTS OF STREPTOMYCES AVERMITILISSTRAINS

A. Spores as Inocula

1. Spore preparations were prepared by standard procedures, the numberof viable spores estimated by plating dilutions on germination agar, andaliquots frozen at -70° C. in 40% glycerol.

2. Before use, spore stocks were centrifuged at 1000 g for 10 minutesand resuspended in an equal volume of 0.85% saline.

3. Approximately 10⁷ spores were inoculated into 30 ml of Modified Yeastextract-Malt extract broth (YEME) medium containing 0.5% glycine (seebelow) in a 300 ml three or four-baffled flask.

B. Frozen Sonicated Mycelia as Inocula

1. Mycelial cultures of PGS-119 were grown in Trypticase Soy Broth (TSB)to a turbidity of 2 to 9 at 600 nm. The culture was homogenized 10 timeswith a glass tissue grinder.

2. The homogenized mycelia were diluted two-fold in TSB and 20 ml wasadded to a sterile polypropylene centrifuge tube. An ultrasonic probewas submerged to a depth of 1 to 2 cm into the liquid, and the samplesonicated at 50% intensity for 10 seconds. Sonication dispersed themycelial masses into single or double cellular units which producedrapid exponential growth when subcultured.

3. Sonicated mycelial preparations were diluted to a final concentrationof 40% glycerol, pipetted into vials, and frozen at -70° C.

4. Aliquots were thawed at room temperature as needed to inoculate intoYEME medium as in Step A.3 above.

C. Colonies on Agar Medium as Inocula

1. Six mature colonies of PGS-119 growing on TSA or YPD-2 agars wereintroduced with a loop into 200 μl of sterile water in a microfuge tube.

2. The mycelial mixture was homogenized with disposable pestle.

3. The homogenized colonies were added to YEME medium as in Step A.3above.

D. Preparation of Protoplasts from Mycelia Grown in Glycine

1. Cultures were incubated in a shaking water bath at 29° C. on setting8 for ca. 65 hours.

2. The mycelia were observed microscopically under 40× phase 2magnification and harvested in a polypropylene centrifuge tube at about1475 g for 10 minutes at 20° C.

3. The supernatant solution was discarded and the mycelial pellet wasre-suspended in 10 ml Protoplast (P) Buffer (see below). The pellet washomogenized 5-10 times with a tissue grinder to disperse clumps.

4. The sample was centrigued at ca. 1000 g for 10 minutes. Thesupernatant solution was discarded and the pellet gently resuspended in10 ml of P buffer.

5. The washing step above was repeated.

6. The mycelial pellet was resuspended in 10 ml of a 1.0 mg/ml freshlysozyme solution in P buffer which had been filter-sterilized bypassage through a 0.22 micron filter.

7. The mycelial mixture was incubated in a water bath at 37° C. withgentle shaking for 60 minutes. The samples were resuspended in thelysozyme solution every 15 minutes. Samples were observedmicroscopically under 40× phase illumination for the presence ofprotoplasts.

8. Mycelial preparations were triturated three times with a 5 ml pipetto free protoplasts from their cell walls.

9. Preparations were filtered through glass wool or non-absorbentcotton.

10. Protoplasts were sedimented by centrifuging at ca. 1000 g for 7minutes, gently resuspended in 5 ml P buffer, and observed under 40×phase magnification.

11. The protoplasts were sedimented as above and resuspended in 1.0 ml Pbuffer. Dilutions of this suspension were made in P buffer and distilledwater and plated on regeneration medium. Colonies which arose fromprotoplast preparations diluted in distilled water were assumed to bederived from incompletely or non-protoplasted mycelial units.

12. Protoplasts were frozen on ice in 200-300 μl aliquots at -70° C.They were removed from the ice 18-24 hours later.

FUSION OF PROTOPLASTS WITH POLYETHYLENE GLYCOL (PEG) 1000

1. All the experiments described herein were performed with a single lotof PEG 1000 (Sigma Chemical Co., St. Louis, Mo. 63178) which producedlittle apparent toxicity in our hands.

2. One (1.0) g aliquots of PEG were autoclaved in glass vials, 1.0 ml ofP buffer was added, and the PEG was dissolved by heating the vial to 55°C. or the PEG was weighed, dissolved in P buffer and filter-sterilizedjust before use. PEG solutions were used at ambient temperatures.

3. Protoplasts were freshly prepared or thawed rapidly from -70° C.stocks under running water. Approximately equal numbers of protoplastsof each genotype were pipetted gently into a polycarbonate centrifugetube. For freshly-prepared protoplast preparations, turbidities weremeasured and several different concentrations were fused. The volume ineach tube was adjusted to 5.0 ml with P buffer.

4. The fusion mixture was centrifuged at ca. 1000 g for 7 minutes.

5. The supernatant solution was decanted carefully. The protoplastpellet was gently resuspended to a final volume of 200 μl with P buffer.

6. Eight hundred (800) μl of 50% PEG was added rapidly to the fusionmixture. The preparation was mixed by drawing it up into a Pasteur pipetand expelling it again. The fusion was incubated for 2 minutes at roomtemperature. Nine (9) ml of P buffer was added to dilute the PEG.Additional fusions were performed serially so that incubation intervalswere accurate.

7. The fusion mixtures were centrifuged as in Step 4 above, thesupernatant solution decanted carefully and the fused, washedprotoplasts were resuspended in 1.0 ml of P buffer.

8. The fusion mixture was serially diluted 10⁻¹ and 10⁻² in P buffer.

9. Fusions of each strain alone were performed in every experiment, andplated as controls.

10. Dilutions of each protoplast preparation (viable counts) were platedto determine numbers of viable regenerants of each strain used in thefusion procedure.

REGENERATION OF PROTOPLASTS

1. Protoplast suspensions, fusion mixtures, or self-fusions were dilutedas appropriate in P buffer and plated in 100 μl aliquots ontoregeneration agar media, using gentle spread technique. Spreading thefused protoplasts in soft agar overlays did not significantly improvetheir regeneration.

2. Where appropriate, the procedure described in D.11 above was used.

3. The regeneration plates were incubated right-side up in sealedplastic bags at 29°-30° C. and ca. humidity.

4. For protoplast fusions in which spectinomycin-resistance was used asa dominant selectable marker, regenerating protoplasts were overlaid at18 hours with 3.5 ml of 100 mcg/ml Spectinomycin in soft agar (seebelow) autoclaved and added at <45° C.

5. Protoplasts were incubated for 7-10 days.

    ______________________________________                                        GROWTH MEDIA, REGENERATION MEDIA, AND                                         PROTOPLAST BUFFER                                                             ______________________________________                                        Complete Regeneration Medium                                                  (Modified from Hopwood, et al. 1985. Genetic                                  Manipulation of Streptomyces: A Laboratory Manual, p.                         235)                                                                          Base solution:                                                                Sucrose                 205    g                                              K.sub.2 SO.sub.4        0.25   g                                              MgCl.sub.2.6H.sub.2 O   10.12  g                                              Glucose                 10     g                                              Difco Casaminoacids     0.1    g                                              Difco Yeast Extract     5.0    g                                              Difco Oatmeal Agar      3.0    g                                              Difco Bacto Agar        22.0   g                                              Distilled water to      955    ml                                             Autoclave for 25 minutes at 121° C.                                    After autoclaving, add sterile stocks of:                                     KH.sub.2 PO.sub.4 (0.5%)                                                                              10     ml                                             CaCl.sub.2.2H.sub.2 O   5      ml                                             L-proline (20%)         15     ml                                             MES buffer (1.0M)       10     ml                                             Trace Element Solution* 2.0    ml                                             NaOH (1N)               3.0    ml                                             Adjust pH to 6.5; bring volume to 1 L.                                        *Trace Element Solution (per Liter):                                          ZnCl.sub.2              40     mg                                             FeCl.sub.3.6H.sub.2 O   200    mg                                             CuCl.sub.2.2H.sub.2 O   10     mg                                             MnCl.sub.2.4H.sub.2 O   10     mg                                             Na.sub.2 B.sub.4 O.sub.7.10H.sub.2 O                                                                  10     mg                                             (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                                                         10     mg                                             Spectinomycin Soft Agar Overlays                                              Complete Regeneration Medium as above except:                                 Agar                    4.10   g                                              Autoclave as above. Cool to 55° C. Add 100 mg                          Spectinomycin. Aliquot in 5 ml volumes in capped                              culture tubes. Refrigerate. Autoclave again just                              before use.                                                                   Modified Protoplast (P) Buffer                                                Base solution:                                                                Sucrose                 205    g                                              K.sub.2 SO.sub.4        0.25   g                                              MgCl.sub.2.6H.sub.2 O   2.02   g                                              Distilled water to      977    ml                                             Autoclave for 25 minutes at 121° C.                                    After autoclaving, add in order to sterile stocks of:                         KH.sub.2 PO.sub.4 (0.5%)                                                                              1      ml                                             Trace Element Solution* 2      ml                                             CaCl.sub.2.2H.sub.2 O (3.68%)                                                                         10     ml                                             MES Buffer (1.0M)       10     ml                                             Adjust pH to 6.5; bring volume to 1 l.                                        *Trace Element solution recipe is above.                                      Modified Yeast Extract-Malt Extract (YEME) Medium                             Base solution:                                                                Difco yeast extract     3      g                                              Difco Bacto-peptone     5      g                                              Difco Bacto malt        3      g                                              extract broth                                                                 Glucose                 10     g                                              Sucrose                 300    g                                              Distilled water to      973    ml                                             Autoclave for 25 minutes at 121°  C.                                   After autoclaving, add:                                                       MgCl.sub.2.6H.sub.2 O (2.5M)                                                                          2      ml                                             Glycine (20%)           25     ml                                             Adjust volume to 1 l.                                                         ______________________________________                                    

DESCRIPTION OF THE DRAWINGS

FIG. 1: UV Tracing (240 nm) versus time (minutes) of HPLC chromatographof solvent fraction from solvent extraction of S. avermitilis I-3 (ATCC53567) cells after growth on fatty acid-free medium (WPM SynA 40:40).Peak at 13.12 is oligomycin A.

FIG. 2: UV Tracing (240 nm) of HPLC chromatograph of solvent fractionfrom solvent extraction of S. avermitilis MA4848 (ATCC 31272) cellsafter growth on fatty acid-free medium (WPM SynA 40:40). Products arenatural avermectins.

FIG. 3: UV Tracing (240 nm) of HPLC chromatograph of solvent fractionfrom solvent extraction of S. avermitilis I-3 (ATCC 53567) cells aftergrowth on medium containing cyclopentylcarboxylic acid (see Example 1).

The attached figures are accurate tracings of HPLC curves of thecompounds indicated.

The compositions of media used in the following Examples are presentedbelow. All molecular weight determinations were obtained by fast atombombardment mass spectrometry performed on a VG Model 7070E massspectrometer using a sample matrix of triethylene glycol with solidsodium chloride. (M+Na)⁺ was determined. Electron impact massspectrometry was performed using a VG Model 7070F mass spectrometer toprovide m/e values only values for the principal fragments are recorded.

    ______________________________________                                                       g/l                                                            ______________________________________                                        AS-7 Medium                                                                   thinned starch.sup.a                                                                           20                                                           Ardamine pH.sup.b                                                                              5                                                            Pharmamedia.sup.c                                                                              15                                                           CaCO.sub.3       2                                                            ______________________________________                                         .sup.a Prepared by hydrolysis of starch by alphaamylase from Bacillus         licheniformis (available from Novo Enzymes, Wilton, CT and sold under the     trademark "Termamyl") to a dextrose eequivalent of 40% ± 5%.               .sup.b From Yeast Products, Inc., Clifton, NJ 07012                           .sup.c From Traders Protein., Memphis, TN 38108                               Adjust pH to 7.2 with NaOH.                                              

    AP-5 Medium                                                                   thinned starch   80                                                           Ardamine pH      5                                                            K.sub.2 HPO.sub.4                                                                              1                                                            MgSO.sub.4.7H.sub.2 O                                                                          1                                                            NaCl             1                                                            CaCO.sub.3       7                                                            FESO.sub.4.7H.sub.2 O                                                                          0.01                                                         MnCl.sub.2.7H.sub.2 O                                                                          0.001                                                        ZnSO.sub.4.7H.sub.2 O                                                                          0.001                                                        P-2000 (antifoam)                                                                              1 ml/l                                                       ______________________________________                                         Adjust pH to 6.9 with 25% NAOH.                                          

    g/l distilled H.sub.2 O                                                       ______________________________________                                        WPM Syn A 40:40                                                               thinned starch   40                                                           Potato soluble starch                                                                          40                                                           glutamic acid    1.0                                                          arginine         0.168                                                        cystine          0.084                                                        histidine        0.069                                                        leucine          0.798                                                        lysine           0.297                                                        methionine       0.108                                                        phenylalanine    0.168                                                        threonine        0.174                                                        tryptophan       0.048                                                        tyrosine         0.192                                                        K.sub.2 HPO.sub.4                                                                              1.0                                                          MgSO.sub.4.7H.sub.2 O                                                                          1.0                                                          NaCl             1.0                                                          CaCO.sub.3       3.5                                                          FeSO.sub.4.7H.sub.2 O                                                                          0.01                                                         MnCl.sub.2.4H.sub.2 O                                                                          0.001                                                        ZnSO.sub.4.7H.sub.2 O                                                                          0.001                                                        ______________________________________                                         pH adjusted to 6.8-7.0, stir 30 minutes at 121° C.                

    WPM Syn B 40:40                                                               potato soluble starch                                                                          40                                                           thinned starch   40                                                           glutamic acid    0.390                                                        arginine         0.168                                                        cystine          0.084                                                        histidine        0.069                                                        lysine HCl       0.297                                                        methionine       0.108                                                        phenylalanine    0.168                                                        threonine        0.174                                                        tryptophan       0.048                                                        tyrosine         0.192                                                        K.sub.2 HPO.sub.4                                                                              1                                                            MgSO.sub.4.7H.sub.2 O                                                                          1                                                            NaCl             1                                                            CACO.sub.3       3.5                                                          FeSO.sub.4.7H.sub.2 O                                                                          0.01                                                         MnCl.sub.2.4H.sub.2 O                                                                          0.001                                                        ZnSO.sub.4.7H.sub.2 O                                                                          0.001                                                        ______________________________________                                         pH adjusted to 6.8-7.0, stir 30 minutes at 121° C.                

General High Performance Liquid Chromatography (HPLC) Procedures

Mobile Phase:

150 ml of water

70 ml of acetonitrile

bring to 1 liter with methanol

Column:

Ultrasphere ODS 25 cm (Beckman Instruments, Fullerton, Calif.92634-3100)

flow: 0.75 ml/minute

detection: UV a 240 nm

attenuation: near 6

Sample diluent (D):

35 ml acetonitrile plus 390 ml methanol

Standards:

1. weigh 0.5 mg avermectin A2A into 10 ml flask and bring to volume withmethanol

2. weigh 0.5 mg test product into 10 ml flask and bring to volume withmethanol

1 and 2 are standard stock solutions; for standard solution to run:

take 100 ul (1) and 100 ul (2) into a vial add 800 ul mobile phase

Samples:

1. Take 1 ml of well shaken broth; spin down

2. Remove as much supernatant as possible without disturbing pellet

3. Add 100 ul of HPLC water to the pellet and vortex mix to disperse

4. Add 2 ml diluent (D) and mix well

5. Filter the same and run on HPLC.

The natural avermectins were subjected to this HPLC chromatographicprocedure and the retention time of the peaks of the individualavermectins divided by the retention time observed for the oligomycin Apresent and which serves as internal standard for a given HPLCdetermination. Oligomycin A is almost always observed by HPLC asby-product of S. avermitilis fermentations and is the only product seenon HPLC produced by the mutants described herein when they are culturedin a medium free of acids RCOOH wherein R is as defined herein or inmedium free of compounds convertible to acids of the formula RCOOHwherein R is as defined herein. Typically, oligomycin A retention timeis 12.5-14 minutes. The ratio of the retention times (RT) affords a moresignificant basis for comparing the identity and yields of avermectinproducts. The general order of appearance of the avermectin products onHPLC is B2, A2, B1 and A1 (FIG. 2).

    ______________________________________                                        Natural                                                                       Avermectin    RT/RT (oligomycin A)                                            ______________________________________                                        B2b           0.70                                                            B2a           0.84                                                            A2b           0.90                                                            A2a           1.09                                                            B1b           1.40                                                            B1a           1.83                                                            A1b           1.83                                                            A1a           2.42                                                            ______________________________________                                        Non-Natural                                                                   Avermectin    RT/RT (oligomycin A)                                            ______________________________________                                        cyclopentyl B2                                                                              0.94                                                            cyclopentyl A2                                                                              1.23                                                            cyclopentyl B1                                                                              1.99                                                            cyclopentyl A1                                                                              2.62                                                            ______________________________________                                    

Ratios were determined from FIG. 2 for the natural avermectins (notethat B1a and A1b are unresolved) and from FIG. 3 for the non-naturalavermectins. Retention times vary 1-2 minutes on different days, witholigomycin A generally appearing near 12.5-14 minutes.

In the following examples the avermectins were determined by the abovedescribed HPLC procedure.

EXAMPLE 1 Cyclopentyl Avermectin A2

S. avermitilis I-3 (ATCC 53567) was cultured at 28°-30° C. in AS-7medium with shaking for 24 hours. A 5 ml portion was used to inoculate a500 ml flask containing 100 ml AS-7 medium and incubation was carriedout under the same conditions for 24 hours; 1 ml of this culture wasused to inoculate AP-5 medium (40 ml in 300 ml flask) to which 24 hourslater was added 0.4 g/l of cyclopentane carboxylic acid (sodium salt).The product flasks were run with shaking at 28°-30° C. By 240 hoursthere was 35 mg/l cyclopentyl avermectin A2 produced while thecorresponding natural A2a titer was 0. Other cyclopentyl avermectinswere also produced.

EXAMPLE 2 Cyclopentyl Avermectin A2

A frozen vial of S. avermitilis HL-026 (ATCC 53568) was used toinoculate 100 ml of AS-7 medium in a 500 ml flask. Growth accompaniedincubation at 28°-30° C. with shaking for 24 hours. A 1 ml aliquot wasused to inoculate two additional 500 ml flasks containing 100 ml of AS-7medium, and these latter flasks, after 18 hours incubation, were used toinoculate 10 liters of AP-5 (less NaCl) medium. After 24 hours ofincubation at 28° C., 0.4 g/l of cyclopentane carboxylic acid was addedto the medium. Agitation was such that dissolved oxygen was maintainedabove 20 percent of saturation. Cyclopentyl A2 titers at 120, 168, 216,264 and 312 hours were 16, 40, 65, 88 and 110 mg/l, respectively. By wayof contrast, the corresponding natural avermectin A2a titer was 0 (i.e.,not detectable) in these samples.

EXAMPLE 3 Cyclopentyl Avermectin A2

In this experiment, the production medium was enriched and multipleadditions of cyclopentane carboxylic acid were made to increasecyclopentyl avermectin titers. The conditions for inoculum developmentand fermentation were the same as those described in Example 2 exceptfor the following: additional 5 g/l Ardamine pH (for a total of 10 g/l)was included in the AP-5 medium and 0.4, 0.2, and 0.2 g/l. ofcyclopentane carboxylic acid were added at 30, 172 and 220,respectively. The cyclopentyl avermectin A2 titers were 1.2, 11, 78, 137and 214 mg/l at 120, 168, 216, 264 and 312 hours, respectively.

EXAMPLE 4 Cyclopentyl Avermectins

A frozen vial of S. avermitilis HL-026 (ATCC 53568) was used toinoculate 100 ml of AS-7 medium in a 500 ml baffled flask which wasincubated for 24-28 hours at 28°-30° C. Then, 1 ml of this culture wasused to inoculate a 300 ml flask containing 40 ml of AP-5 (less NaCl butplus 0.6 g/l glutamic acid) medium. After 96 hours of incubation at28°-30° C. with shaking, 0.4 g/l of cyclopentane carboxylic acid (sodiumsalt) was added. HPLC chromatography of a 216 hour sample showedcyclopentyl avermectins B2, A2, B1 and A1 present with retention timesof 12.32, 15.86, 25.28 and 32.96 minutes, respectively.

EXAMPLE 5 Cyclopentyl Avermectin A2

In this example, S. avermitilis I-3 (ATCC 53567) and S. avermitilisHL-026 (ATCC 53568) were grown under identical conditions. Three media(AP-5, WPM Syn A 40:40, and WPM Syn B 40:40) were used. A frozen cultureof each organism was used to inoculate 100 ml of AS-7 medium in 500 mlbaffled flasks, which were incubated subsequently for 24-26 hours at28°-30° C. Then, 1 ml of each culture was used to inoculate 300 mlflasks, each flask containing 40 ml of one of the three media.Duplicates of each flask were run. After 24 hours of incubation at 28°C. with shaking, each flask received 0.4 g/l of cyclopentylcarboxylicacid (sodium salt), and after a total of 192 hours of incubation, thetiters of the principle product, cyclopentyl avermectin A2, weredetermined (Table I).

                  TABLE I                                                         ______________________________________                                                                  Cyclopentyl                                                        Strain of S.                                                                             avermectin A2                                       Medium         avermitilis                                                                              mg/l                                                ______________________________________                                        AP-5           ATCC 53567 29                                                                 ATCC 53568 67                                                  WPM Syn A 40:40                                                                              ATCC 53567 35                                                                 ATCC 53568 115                                                 WPM Syn B 40:40                                                                              ATCC 53567 38                                                                 ATCC 53568 36                                                  ______________________________________                                    

EXAMPLE 6 Cyclohexyl Avermectins

In this example, 0.2 g/l of cyclohexane carboxylic acid was added at 96hours instead of cyclopentane carboxylic acid, and all other conditionswere the same as those described in Example 4. Four cyclohexylavermectins were identified on the HPLC chromatogram of a 240 hoursample. The retention times for cyclohexyl avermectins B2, A2, B1 and A1were 14.84, 19.26, 31.46 and 41.14 minutes, respectively.

EXAMPLE 7 3-Cyclohexenyl Avermectins

In this example, 0.2 g/l of 3-cyclohexene carboxylic acid was added at96 hours instead of cyclopentane carboxylic acid, and all otherconditions were the same as those described in Example 4. Severalcyclohexenyl avermectins were identified on the HPLC chromatogram of a312 hour sample. Their retention times are 12.88 (B2), 16.39 (A2),27.37/28.36 (B1 isomers) and 35.80/37.13 (A1 isomers) minutes,respectively.

EXAMPLE 8 3-Thienyl Avermectins

In this example, 0.05 g/l of thiophene-3-carboxylic acid was added at 96hours instead of cyclopentane carboxylic acid, and all other conditionswere the same as those described in Example 4. Four 3-thienylavermectins were identified on the HPLC chromatogram of a 312 hoursample. The retention times for 3-thienyl avermectins B2, A2, B1 and A1were 6.96, 8.76, 13.8 and 23.5 minutes, respectively.

EXAMPLE 9 1-Methylthioethyl Avermectins

In this example, 0.4 and 0.2 g/l of 2-methylthiopropionic acid wereadded at 24 and 96 hours, respectively, instead of cyclopentanecarboxylic acid, and all other conditions were the same as thosedescribed in Example 4. Two 1-methylthioethyl avermectins wereidentified on the HPLC chromatogram of a 240 hour sample. The retentiontimes for 1-methylthioethyl avermectins A2 and B1 were 9.30 and 13.06minutes, respectively. The peak, with an estimated retention time ofabout 7.2 minutes, emerging on the front shoulder of the 7.557 minutepeak is believed to be the B2 compound, and the A1 compound is believedto be under the 17.22 minute peak.

EXAMPLE 10 2-pentyl Avermectins

In this example, 0.2 g/l of 2-methylvaleric acid was added at 96 hoursinstead of cyclopentane carboxylic acid, and all other conditions werethe same as those described in Example 4. Four 2-pentyl avermectins wereidentified on the HPLC chromatogram of a 312 hour sample. The retentiontimes for 2-pentyl avermectins B2, A2, B1 and A1 were 12.88, 16.58,31.90 and 41.92 minutes, respectively.

EXAMPLE 11 1-Methyl-3-butenyl Avermectins

In this example, 0.2 g/l of 2-methyl-4-pentenoic acid was added at 96hours instead of cyclopentane carboxylic acid, and all other conditionswere the same as those described in Example 4. Four 1-methyl-3-butenylavermectins were identified on the HPLC chromatogram of a 312 hoursample. The retention times for 1-methyl-3-butenyl avermectins B2, A2,B1 and A1 were 11.13, 14.78, 22.10 and 28.92 minutes, respectively.

EXAMPLE 12 1-Methyl-1-butenyl Avermectins

In this example, 0.2 g/l of 2-methyl-2-pentenoic acid was added at 96hours instead of cyclopentane carboxylic acid, and all other conditionswere the same as those described in Example 4. Four 1-methyl-1-butenylavermectins were identified on the HPLC chromatogram of a 312 hoursample. The retention times for 1-methyl-1-butenyl avermectins B2, A2,B1 and A1 were 11.59, 14.93, 25.29 and 33.18 minutes, respectively.

EXAMPLE 13

In this example, the use of the mutant to prepare the naturalavermectins derived from L-valine in the absence of the avermectinsderived from L-isoleucine is demonstrated. The contents of a frozen vialcontaining S. avermitilis I-3 (ATCC 53567) was transferred to a 500 mlbaffled flask containing 100 ml of AS-7 medium. After approximately oneday at 28°-30° C. with shaking (ca. 200 rpm), 1 ml of the culture isused to inoculate 40 ml of WPM Syn A 40:40 medium in a 300 ml flask,which is subsequently incubated at 28°-30° C. for 24 hours with shaking.At this time, 4 ml of a filter-sterilized solution of isobutyric acid(neutralized to pH 6-7 with NaOH), 4 mg/ml is added and incubation wascontinued as above for a total of 8 days. HPLC analysis showed 4 majorpeaks (excluding oligomycin). (In similar experiments with2-methylbutyric acid replacing isobutyric acid, the complementary 4peaks of avermectins derived from L-isoleucine were seen.

EXAMPLE 14

The procedure of Example 1 was repeated but substituting the primercompounds listed below for cyclopentane carboxylic acid. The avermectins(formula I compounds wherein R² is the oleandrose disaccharide moietyand R, R¹ and R³ are as shown) identified from a given fermentation arealso listed.

    __________________________________________________________________________                                Product:                                                                      RT/RT (oligomycin A)                              Cpd                                                                              Primer Compound                                                                              R         B.sub.2                                                                          A.sub.2                                                                          B.sub.1                                                                          A.sub.1                                  __________________________________________________________________________     1 2-methylvaleric acid                                                                         pent-2-yl 1  1.287                                                                            2.478                                                                            3.255                                     2 2-methylpent-4-enoic acid                                                                    4-penten-2-yl                                                                           0.853                                                                            1.090                                                                            1.694                                                                            2.217                                                                0.904                                                                            1.133                                                                            1.784                                                                            2.346                                     3 1-cyclohexene carboxylic                                                      acid           cyclohexen-l-yl                                                                         0.785                                                                            1.021                                                                            1.665                                                                            2.179                                     4 thiophene-2-carboxylic                                                                       thien-2-yl   0.694                                                                            1.143                                                                            1.499                                       acid                                                                        5 3-furoic acid  3-furyl      0.705                                                                            1.095                                        6 cyclobutane carboxylic acid                                                                  cyclobutyl                                                                              0.728                                                                            0.933                                                                            1.546                                                                            2.027                                     7 cyclopentane carboxylic acid                                                                 cyclopentyl                                                                             0.960                                                                            1.236                                                                            1.970                                                                            2.568                                     8 cyclohexane carboxylic acid                                                                  cyclohexyl                                                                              1.206                                                                            1.565                                                                            2.556                                                                            3.343                                     9 cycloheptane carboxylic                                                                      cycloheptyl                                                                             1.465                                                                            1.923                                             acid                                                                       10 3-cyclohexene-1-carboxylic                                                                   cyclohex-3-enyl                                                                         1  1.273                                                                            2.125                                                                            2.780                                       acid                                                                       11 2-methylthiopropionic                                                                        1-methylthioethyl                                                                       0.565                                                                            0.730                                                                            1.025                                                                            1.351                                       acid                                                                       12 thiophene-3-carboxyic acid                                                                   thien-3-yl                                                                              0.539                                                                            0.639                                                                            1.069                                                                            1.388                                    13 hydroxymethylcyclopentane                                                                    cyclopentyl                                                                             identical to 7                                    14 3-thiophene carboxaldehyde                                                                   thien-3-yl                                                                               identical to 12                                  15 3-cyclohexylpropionic acid                                                                   cyclohexyl                                                                              identical to 8                                    16 3-cyclopentylpropionic acid                                                                  cyclopentyl                                                                             identical to 7                                    17 hydroxymethylcyclobutane                                                                     cyclobutyl                                                                              identical to 6                                    18 3-cyclopentyl-l-propanol                                                                     cyclopentyl                                                                             identical to 7                                    19 cyclobutylmethylamine                                                                        cyclobutyl                                                                              identical to 6                                    20 ethyl cyclobutane-                                                            carboxylate    cyclobutyl                                                                              identical to 6                                    21 2-(cyclobutylcarbonyl)-                                                       propionic acid cyclobutyl                                                                              identical to 6                                    22 ethyl 2-(3-thiophene                                                          carbonyl)propionate                                                                          thien-3-yl                                                                               identical to 12                                  23 1-methylcyclopropane                                                                         1-methylcyclopropyl                                                                        1.236                                             carboxylic acid                                                            24 2-methylpent-2-enoic                                                                         2-penten-2-yl                                                                           0.812                                                                            1.091                                                                            1.923                                                                            2.523                                       acid                     0.882                                                                            1.135                                          25 2-furoic acid  2-furyl      0.709                                                                            1.146                                       26 5-methylthiophene-2-                                                                         5-methylthien-2-yl                                                                      0.533    1.514                                       carboxylic acid                                                            27 1-methylcyclopropane                                                                         1-methylcyclopropyl                                                                        1.236                                             carboxylic acid                                                            28 cyclopropane carboxylic                                                                      cyclopropyl                                                                             0.802                                                                            1.048 2.236                                       acid                                                                       __________________________________________________________________________

Other physico-chemical data for certain of the above compounds arepresented below.

    ______________________________________                                        Cpd       Physico-Chemical Data                                               ______________________________________                                        6 (A2)    white powder; m.p. 135-140° C.; molecular                              weight = 925; m/e 596, 454, 321, 303, 275,                                    237, 219, 209, 191, 179, 167, 145, 127,                                       113, 111, 95 and 87.                                                6 (A1)    white powder; m.p. 120-124° C.; molecular                              weight = 907; m/e 578, 303, 275, 257, 219,                                    191, 167, 145, 127, 113, 111, 95 and 87.                            6 (B2)    white powder; m.p. 110-112° C.; molecular                              weight = 911; m/e 321, 303, 261, 257, 237,                                    219, 209, 191, 179, 167, 145, 127, 113,                                       111, 95 and 87.                                                     6 (B1)    white powder; m.p. 135-138° C.; molecular                              weight = 893; m/e 303, 261, 257, 219, 191,                                    167, 145, 127, 113, 111, 95 and 87.                                 8 (A2)    white powder; m.p. 112-117° C.; molecular                              weight = 953; m/e 624, 482, 349, 349, 331,                                    275, 265, 247, 237, 219, 207, 195, 179,                                       145, 127, 113, 111, 95 and 87.                                      10 (A2)   white powder; m.p. 131-135° C.; molecular                              weight = 951; m/e 624, 480, 347, 329, 275,                                    263, 245, 235, 217, 205, 193, 179, 145,                                       127, 113, 111, 95 and 87.                                           12 (A2)   white powder; m.p. 167° C.; molecular                                  weight = 953; m/e 349, 331, 275, 265, 257,                                    247, 237, 219, 195, 145, 127, 113, 95                                         and 87.                                                             ______________________________________                                    

EXAMPLE 15

Repetition of the procedure of Example 14, but using the primercompounds listed below affords the corresponding avermectin products: Ravermectins A1, A2, B1 and B2 wherein R corresponds to the R group ofthe primer compound.

    ______________________________________                                        Primer Compound     R                                                         ______________________________________                                        2,3-dimethylbutyric 1,2-dimethylpropyl                                        2-methylhexanoic acid                                                                             hex-2-yl                                                  2-cyclopropyl propionic                                                                           1-cyclopropylethyl                                        acid                                                                          4,4-difluorocyclohexane                                                                           4,4-difluoro-                                             carboxylic acid     cyclohexyl                                                4-methylenecyclohexane                                                                            4-methylene-                                              carboxylic acid     cyclohexyl                                                3-methylcyclohexane 2-methylcyclohexyl                                        carboxylic acid                                                               (cis/trans)                                                                   1-cyclopentene carboxylic                                                                         cyclopenten-1-yl                                          acid                                                                          tetrahydropyran-4-  tetrahydropyran-4-yl                                      carboxylic acid                                                               2-chlorothiophene-4-                                                                              2-chlorothien-4-yl                                        carboxylic acid                                                               (S)-2-methylpentanoic acid                                                                        (S)-pent-2-yl                                             (R)-2-methylpentanoic acid                                                                        (R)-pent-2-yl                                             2-methylcyclopropane                                                                              2-methylcyclopropyl                                       carboxylic acid                                                               2-methyl-4-methoxybutyric                                                                         4-methoxybut-2-yl                                         acid                                                                          tetrahydrothiophene-3-                                                                            tetrahydrothien-3-yl                                      carboxylic acid                                                               3-methylcyclobutane 3-methylcyclobutyl                                        carboxylic acid                                                               3-fluorocyclobutane 3-fluorocyclobutyl                                        carboxylic acid                                                               3-methylene cyclobutane                                                                           3-methylene-                                              carboxylic acid     cyclobutyl                                                2-methyl-4-methylthio-                                                                            4-methylthiobut-2-yl                                      butanoic acid                                                                 tetrahydrothiopyran-4-                                                                            tetrahydrothio-                                           carboxylic acid     pyran-4-yl                                                3-cyclopentenemethanol                                                                            cyclopent-3-enyl                                          ______________________________________                                    

EXAMPLE 16 Cyclopentyl Avermectin A2 Recovery

This example is given to demonstrate a recovery process for the A2-likeavermectins formed from the cyclopentanecarboxylic acid precursor. Wholebroth (from a fermentation similar to that of Example 2) was filtered,and the mycelial cell mass was extracted twice with acetone (3 volumes).The acetone extract was concentrated to an aqueous oil, the oilextracted with methylene chloride and the methylene chloride solutionconcentrated to a dark brown oil. The dark brown oil was then dissolvedin methanol/water (4:1) and the resulting solution extracted with hexaneto remove fatty acids/lipids. Evaporation of the methanol/water affordeda light brown oil containing approximately 10% cyclopentyl avermectin A2w/w. The crude avermectin oil was then diluted with chloroform (3 ml ofCHCl₃ per g of oil), activated charcoal (0.35 g/g of oil) and silica gel(1 g/g of oil) were added, and the mixture stirred for an hour and thenfiltered. The filtrate was concentrated, and the resulting oil dilutedwith isopropyl ether (1 ml of IPE per g of oil). The resulting solutionwas then gradually dripped into a large volume of hexane (25 ml ofhexane/g of oil), whereupon a white, crude avermectin powderprecipitated. The first crop was isolated via filtration and thefiltrate was then cooled to approximately 5° C. to precipitate out asecond crop.

The crudes were further processed by preparative high performance liquidchromatography to obtain purified product. Crudes were dissolved (4.1ml. solvent per gram of crude powder) in 25/25/25/50/0.125 IPE/CH₃CN/ethyl acetate/hexane/acetic acid. Samples were injected onto a 41.4mm by 25 cm silica preparative column and eluted with the above solventat 30 ml/minute and the product-containing peak collected. The collectedfractions were concentrated and diluted with MeOH/H₂ O (86/14) such that1 ml contained approximately 50 mg product. Samples were then injectedon a C-18 preparative column (same dimensions as silica column) andeluted at 18-20 ml/minute with MeOH/H₂ O (275 ml H₂ O to 2 liters withMeOH). The fractions were again concentrated and passed over the C-18prep column a second time. Evaporation of the product-containingfractions to dryness afforded pure title product.

The corresponding cyclopentyl avermectin B2, A1 and B1 are recovered bycollecting the appropriate fractions from the above described HPLCsteps.

EXAMPLE 17

S. avermitilis JC 923 (ATCC 53669) mycelium from a YPD-2 agar medium wasused to inoculate 50 ml of AS-7 medium in a 300 ml baffled flask, whichwas maintained with shaking (220 rpm) at 30° C. for 24 hours. Then, eachof two 300 ml flasks (no baffles) containing 50 ml of AP-5 medium wasinoculated with one ml of the culture. One flask contained2-methylbutyric acid (0.1%) and the other no 2-methylbutyric acid.Fermentation was carried out at 30° C. for 11 days with shaking. Thecontents of each flask was worked up in the same way. The whole brothwas extracted with a four-fold volume excess of acetonitrile:methanol(810:75). After vigorous shaking to promote antibiotic extraction fromthe cells, the clarified supernatant was analyzed via HPLC foravermectins. When no fatty acid precursor was added, no detectableavermectins ("a" type) were found (sensitivity ≦0.20 mg/L). In thepresence of the 2-methylbutyric acid avermectins B2a, A2a, B1a, A1a weremeasured at 0.5, 1.3, 1.4 and 1.1 mg/L, respectively.

EXAMPLE 18

In this example, AP-5 production fermentations of S. avermitilis JC 923(ATCC 53669) were prepared from AS-7 grown inocula as in Example 17. Theprimer compound 2-methylbutyric acid was present at 0.05% concentration.In this case, the unsupplemented control fermentation (no fatty acidprecursor) gave values of 0.3, 0.9, <0.2 and <0.2 mg/L for avermectinsB2a, A2a, B1a and A1a, respectively, whereas the results from the fattyacid supplemented fermentations were 2.8, 5.0, 4.5 and 2.3 mg/L,respectively.

EXAMPLE 19

In this example, AP-5 production fermentations of S. avermitilis JC 923(ATCC 53669) were prepared from AS-7 inocula as in Example 18. In thisexample, 2-methylbutyric acid was added at 0.05% concentration 48 hoursafter the inoculation of the AP-5 medium, and cells were harvested byfiltration, weighed (wet weight) and extracted with a four-fold weightexcess of extraction solvent. This concentration step enable greatersensitivity in avermectin titer measurements over the previous cases inwhich whole broths were directly extracted. In the present example,levels of B2a, A2a, B1a, A1a of 2.5, 8.5, 4.5 and 3.5 mg/L weredetermined for the 2-methylbutyric acid supplemented fermentation, ascompared to values of 0.3, 0.3, 0.3 and 0.1 for the unsupplementedcontrol. These latter, low levels are presumably attributable to lowlevels of endogenous fatty acid compounds in the crude AP-5 productionmedium.

EXAMPLE 20

Fermentations were carried out as in Example 19, except thatcyclopentane carboxylic acid at 0.045% concentration was substituted forthe 2-methylbutyric acid supplementation. Cyclopentylavermectin A2 wasdetermined to be present at 4 mg/L concentration.

EXAMPLE 21

Fermentations (16) of S. avermitilis PGS-119 (ATCC 53670) were carriedout in 50 ml of AP-5 medium in 300 ml flasks (no baffles) incubated at30° C. The medium was inoculated with one ml of a 24 hour culture of thestrain in AS-7 medium, 30° C. incubation, 50 ml medium in 300 baffledflask. After 66 hours of growth in the AP-5 medium, isobutyric acid at aconcentration of 0.1% was added to 8 of the flasks. Using the work-upprocedure of Example 17, these supplemented flasks yieldedconcentrations of B2b, A2b, B1b and A1b equal to (average of twoexperiments) 5.6, 45, 45 and 68 mg/L, respectively. The unsupplementedcultures gave values of 0.5, 4.0, 4.5 and ≦8.5 mg/L, respectively. Inaddition, for the latter fermentation, values of 1.1, 1.1, undeterminedand <0.2 mg/L for the corresponding B2a, A2a, B1a and A1a avermectinswere found.

EXAMPLE 22

In this example, four AP-5 fermentations of S. avermitilis PGS-119 (ATCC53670) were carried out in 2 ml cultures in plastic tubes (15 ml).Inocula were prepared as described in Example 21 in AS-7 medium, at 30°with shaking of tubes maintained in a 30° slanted position. The fattyacid precursor, cyclohexane carboxylic acid (CHC), at a 0.045%concentration was added at 96 hours after AP-5 inoculation to two of thetubes. Four hundred hours following AP-5 inoculation, with 0.05 ml ofthe AS-7 culture, 8 ml of extraction solvent (acetonitrile:methanol(810:75)) was added to every tube and avermectin titers were determinedin the supernatants by HPLC analysis as described in the text. Foravermectins CHC-B2, CHC-A2, CHC-B1, CHC-A1, A2b, B1b, A1b, A2a and A1a,concentrations were equal to (average of two tubes) 3.5, 6.2, 3.0, 1.4,0.2, 0.2, 0.4, 0.2, <0.2 mg/L, respectively The corresponding values forthe two fermentation tubes receiving no precursor acid were <0.2, <0.2,<0.2, <0.2, 4.2, 2.9 9.3, 1.2, 0.3 mg/L, respectively. All other naturalavermectins were essentially undetectable.

EXAMPLE 23

The procedure of Example 17 was repeated, but using the primer compoundor precursor thereto listed below in place of 2-methylbutyric acid. Ineach instance, non-natural avermectins RA1, RA2, RB1 and RB2 wherein the25-substituent corresponds to the R group of the primer compound orprecursor thereto are produced.

Primer Compound

cyclohexane carboxylic acid

cyclobutane carboxylic acid

2-methylpent-4-enoic acid

3-cyclohexene-1-carboxylic acid

2-methylthiopropionic acid

3-furoic acid

thiophene-3-carboxylic acid

1-methylcyclopropane carboxylic acid

3-cyclopentylpropionic acid

ethylcyclopentane carboxylate

tetrahydrothiophene-3-carboxylic acid

3-cyclopentenemethylamine

cyclobutylmethyl amine

4-hydroxymethylcyclopentene

cyclopentane carboxamide

2-furaldehyde

EXAMPLE 24

Repetition of the procedure of Example 22, but using the primercompounds and precursors listed below in place of cyclohexane carboxylicacid affords the corresponding R-avermectins A1, A2, B1 and B 2,respectively.

Primer Compound

(S)-2-methylpentanoic acid

2-chlorothiophene-4-carboxylic acid

3-thiophenecarboxylic acid

2,3-dimethylbutyric acid

2-methylthiopropionic acid

2-furaldehyde

2-methyl-4-methoxybutyric acid

2-(cyclobutylcarbonyl)propionic acid

We claim:
 1. A biologically pure culture of Streptomyces avermitiliswhich produces natural avermectins having an isopropyl or a(S)-sec-butyl moiety at C-25 upon fermentation in an aqueous nutrientmedium comprising an assimilable source of carbon, nitrogen andinorganic salts under aerobic conditions only when said medium comprisesan acid of the formula R--COOH, or a precursor of said acid of theformula R--(CH₂)_(n) --Z or R--CO--Z wherein n is 0, 2, 4 or 6 and Z is--CH₂ OH, --CHO, --CH₂ NH₂, --COOR⁵ or --CONHR⁶ wherein R⁵ is H or (C₁-C₆)alkyl and R⁶ is H, (C₁ -C₄)alkyl, --CH(COOH)CH₂ COOH,--CH(COOH)(CH₂)₂ COOH or --CH(COOH)(CH₂)₂ SCH₃ wherein R is isopropyl or(S)-sec-butyl.
 2. A mutant strain of Streptomyces avermitilis whichproduces natural avermectins having an isopropyl or a (S)-sec-butylmoiety at C-25 upon fermentation in an aqueous nutrient mediumcomprising an assimilable source of carbon, nitrogen and inorganic saltsunder aerobic conditions only when said medium comprises an acid of theformula R--COOH, or a precursor of said acid of the formula R--(CH₂)_(n)--Z or R--CO--Z wherein n is 0, 2, 4 or 6 and Z is --CH₂ OH, --CHO,--CH₂ NH₂, --COOR⁵ or --CONHR⁶ wherein R⁵ is H or (C₁ -C₆)alkyl and R⁶is H, (C₁ -C₄)alkyl, --CH(COOH)CH₂ COOH, --CH(COOH)(CH₂)₂ COOH or--CH(COOH)(CH₂)₂ SCH₃ wherein R is is isopropyl or (S)-sec-butyl;wherein said strain is obtained by a process which comprises mutatingStreptomyces avermitilis ATCC 31271, ATCC 31272 or an avermectinproducing mutant thereof and selecting a mutant whichlacksbranched-chain amino acid transaminase activity.
 3. Streptomycesavermitilis lacking one or both of the following activities:(a)branched-chain 2-oxo-acid dehydrogenase activity wherein permeabilizedcells thereof are unable to produce ¹⁴ CO₂ from added [¹⁴C-1]-2-oxoisocaproic acid, (b) branched-chain amino acid transaminaseactivity wherein said S. avermitilis is unable to grow on media lackingL-iso-leucine, L-leucine and L-valine; and which S. avermitllis producesnatural avermectins having an isopropyl or a (S)-sec-butyl moiety atC-25 upon fermentation in an aqueous nutrient medium comprising anassimilable source of carbon, nitrogen and inorganic salts under aerobicconditions when said medium comprises an acid of the formula R--COOHwherein R is isopropyl or (S)-sec-butyl.
 4. Streptomyces avermitilisaccording to claim 1, said S. avermitilis lacking branched-chain aminoacid transaminase activity.
 5. Streptomyces avermitilis according toclaim 1, said S. avermitilis lacking branched-chain 2-oxo aciddehydrogenase activity.
 6. Streptomyces avermitilis according to claim1, said S. avermitilis lacking branched-chain amino acid transaminaseactivity and branched-chain 2-oxo acid dehydrogenase activity.
 7. Abiologically pure culture of Streptomyces avermitilis which producesnatural avermectins having an isopropyl or a (S)-sec-butyl moiety atC-25 upon fermentation in an aqueous nutrient medium comprising anassimilable source of carbon, nitrogen and inorganic salts under aerobicconditions only when said medium comprises an acid of the formulaR--COOH, or a precursor of said acid of the formula R--(CH₂)_(n) --Zwherein n is 0, 2, 4 or 6 and Z is --CH₂ OH, --CHO, --CH₂ NH₂, --COOR⁵or --CONHR⁶ wherein R⁵ is H or (C₁ -C₆)alkyl and R⁶ is H, (C₁ -C₄)alkyl,--CH(COOH)CH₂ COOH, --CH(COOH)(CH₂)₂ COOH or --CH(COOH)(CH₂)₂ SC₃wherein R is isopropyl or (S)-sec-butyl.
 8. A mutant strain ofStreptomyces avermitilis which produces natural avermectine having anisopropyl or a (S)-sec-butyl moiety at C-25 upon fermentation in anaqueous nutrient medium comprising an assimilable source of carbon,nitrogen and inorganic salts under aerobic conditions only when saidmedium comprises an acid of the formula R--COOH, or a precursor of saidacid of the formula R--(CH₂)_(n) --Z wherein n is 0, 2, 4 or 6 and Z is--CH₂ OH, --CHO, --CH₂ NH₂, --COOR⁵ or --CONHR⁶ wherein R⁵ is H or (C₁-C₆)alkyl and R⁶ is H, (C₁ -C₄)alkyl, --CH(COOH)CH₂ COOH,--CH(COOH)(CH₂)₂ COOH or --CH(COOH)(CH₂)₂ SCH₃ wherein R is isopropyl or(S)-sec-butyl; wherein said strain is obtained by a process whichcomprises mutating Streptomyaes avermitilis ATCC 31271, ATCC 31272 or anavermectin producing mutant thereof and selecting a mutant which lacksbranched-chain 2-oxo-acid dehydrogenase activity or both branched-chain2-oxo-acid dehydrogenase activity and branched-chain amino acidtransaminase activity.